EQUINE ANTIBODY MUTANTS

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Patent Application 63/374,376, filed on Sep. 2, 2022, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates generally to equine antibody mutants and uses thereof. Specifically, the invention relates to one or more mutations in the Fc constant region of equine antibody for improving various characteristics.

BACKGROUND OF THE INVENTION

Equine IgG monoclonal antibodies (mAbs) can be effective therapeutics in veterinary medicine. Several years ago, seven equine IgG subclasses were identified. However, only a limited work has been done to improve the characteristics of equine IgGs.

Through a recycling mechanism, the neonatal Fc receptor (FcRn) prolongs the half-life of an IgG in a pH-dependent interaction with its fragment crystallizable (Fc) region. Specifically, the Fc region spanning the interface of CH2 and CH3 domains interacts with the FcRn on the surface of cells to regulate IgG homeostasis. This interaction is favored by an acidic interaction after IgG pinocytosis and thus IgG is protected from degradation. The endocytosed IgG is then recycled back to the cell surface and released into the blood stream at a slightly alkaline pH thereby maintaining sufficient serum IgG for proper function. Accordingly, the pharmacokinetic profile of IgGs depend on the structural and functional properties of their Fc regions.

Engineering Fc regions to tune their interactions with FcRn has emerged as a promising approach for enhancing the activity of therapeutic antibodies.

Accordingly, there exists a need for novel equine IgG Fc region mutations to improve various characteristics of equine IgGs.

SUMMARY OF THE INVENTION

The invention relates to mutant equine IgGs that exhibit desired characteristics, relative to wild-type equine IgGs. Specifically, the inventors of the instant application have found that substituting an amino acid residue at position 252, 286, 311, 312, 426, 428, 434, or 436 (numbered according to the Eu index as in Kabat) with another amino acid surprisingly and unexpectedly exhibited a desired effect. In an exemplary embodiment, the unexpected desired effects include, but not limited to, enhanced affinity to FcRn.

In one aspect, the invention provides a modified IgG comprising: an equine IgG constant domain comprising at least one amino acid substitution relative to a wild-type equine IgG constant domain, wherein said substitution is at amino acid residue 252, 286, 311, 312, 426, 428, 434, or 436.

In one exemplary embodiment, the equine IgG constant domain is an IgG1 constant domain that comprises one or more of mutations of M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, G426A, G426C, G426D, G426E, G426F, G426H, G426I, G426K, G426L, G426M, G426N, G426P, G426Q, G426R, G426S, G426T, G426V, G426W, G426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

In another aspect, the invention provides a polypeptide comprising: an equine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.

In yet another aspect, the invention provides an antibody or a molecule comprising: an equine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.

In a further aspect, the invention provides a method for producing or manufacturing an antibody or a molecule, the method comprising: providing a vector or a host cell having a nucleic acid sequence that encodes an antibody, wherein said antibody comprises an equine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.

Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 illustrates domain structure of IgG.

FIG. 2 shows the alignment of the amino acid sequences of human IgG1 and equine IgGs 1-7. CH1, hinge, CH2, and CH3 domains are as follows: CH1: residues 118-215; hinge: 216-230; CH2: 231-340; CH3: 341-447. The amino acid residues are numbered according to the Eu index as in Kabat.

FIG. 3. Protein modeling of Equine FcRn and IgG1 Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

FIG. 4. Protein modeling of Equine FcRn, IgG4a-Fc and IgG4b-Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

FIG. 5. Protein modeling of Equine FcRn, IgG7a-Fc and IgG7b-Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

FIG. 6. Root Mean Square Deviation (RMSD) comparisons of wild-type constructs of equine IgG1, IgG4a, IgG4b, IgG7a and IgG7b.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

• • SEQ ID NO.: 1 refers to the amino acid sequence of equine IgG1 Wildtype.
  • SEQ ID NO.: 2 refers to the amino acid sequence of equine IgG2 Wildtype.
  • SEQ ID NO.: 3 refers to the amino acid sequence of equine IgG3 Wildtype.
  • SEQ ID NO.: 4 refers to the amino acid sequence of equine IgG4 Wildtype.
  • SEQ ID NO.: 5 refers to the amino acid sequence of equine IgG5 Wildtype.
  • SEQ ID NO.: 6 refers to the amino acid sequence of equine IgG6 Wildtype.
  • SEQ ID NO.: 7 refers to the amino acid sequence of equine IgG7 Wildtype.
  • SEQ ID NO.: 8 refers to the amino acid sequence of human IgG1.

DETAILED DESCRIPTION OF THE INVENTION

The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

Definitions

In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a molecule” or “a compound” is a reference to one or more of such molecules or compounds and equivalents thereof known to those skilled in the art, and so forth. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

In the specification and claims, the numbering of the amino acid residues in an immunoglobulin heavy chain is that of the Eu index as in Kabat, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The “Eu index as in Kabat” refers to the residue numbering of the IgG antibody and is reflected herein in FIG. 2.

The term “isolated” when used in relation to a nucleic acid is a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is in a form or setting different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide encoded therein where, for example, the nucleic acid molecule is in a plasmid or a chromosomal location different from that of natural cells. The isolated nucleic acid may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand, but may contain both the sense and anti-sense strands (i.e., may be double-stranded).

A nucleic acid molecule is “operably linked” or “operably attached” when it is placed into a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of nucleic acid if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence of nucleic acid if it is positioned so as to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof which does not, as it exists in nature, comprise an Fc region) if it is positioned such that the expressed fusion protein comprises the heterologous protein or functional fragment thereof adjoined either upstream or downstream to the variant Fc region polypeptide; the heterologous protein may by immediately adjacent to the variant Fc region polypeptide or may be separated therefrom by a linker sequence of any length and composition. Likewise, a polypeptide (used synonymously herein with “protein”) molecule is “operably linked” or “operably attached” when it is placed into a functional relationship with another polypeptide.

As used herein the term “functional fragment” when in reference to a polypeptide or protein (e.g., a variant Fc region, or a monoclonal antibody) refers to fragments of that protein which retain at least one function of the full-length polypeptide. The fragments may range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. A functional fragment of a variant Fc region polypeptide of the present invention retains at least one “amino acid substitution” as herein defined. A functional fragment of a variant Fc region polypeptide retains at least one function known in the art to be associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, down regulation of cell surface receptors or may, e.g., increase the in vivo or in vitro half-life of a polypeptide to which it is operably attached).

The term “purified” or “purify” refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody may be purified by complete or substantial removal (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably at least 96%, 97%, 98% or 99%) of at least one contaminating non-immunoglobulin protein; it may also be purified by the removal of immunoglobulin protein that does not bind to the same antigen. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind a particular antigen results in an increase in the percent of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in bacterial host cells is purified by the complete or substantial removal of host cell proteins; the percent of the polypeptide is thereby increased in the sample.

The term “native” as it refers to a polypeptide (e.g., Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of the polypeptide as it commonly occurs in nature or a naturally occurring polymorphism thereof. A native polypeptide (e.g., native Fc region) may be produced by recombinant means or may be isolated from a naturally occurring source.

The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.

As used herein, the term “host cell” refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in situ, or in vivo

As used herein, the term “Fc region” refers to a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain might vary, the equine IgG heavy chain Fc region is usually defined to stretch, for example, from residue 231 to the c-terminus in FIG. 2. In some embodiments, variants comprise only portions of the Fc region and can include or not include the carboxy-terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants without such constant domains (or with only portions of such constant domains) are contemplated.

The “CH2 domain” of an equine IgG Fc region refers to, for example, the residues starting at residue 231 and extending to residue 340 in FIG. 2. The CH2 domain is unique in that it is not closely paired with another domain.

The “CH3 domain” of an equine IgG Fc region generally is the stretch of residues C-terminal to a CH2 domain in an Fc region, for example, residue 341 to the c-terminus in FIG. 2.

A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be operably linked to a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assay, ADCC assays, CDC assays, ADCP assays, target cell depletion from whole or fractionated blood samples, etc.).

A “native sequence Fc region” or “wild type Fc region” refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region commonly found in nature. Exemplary native sequence equine Fc regions are from residue 231 to the c-terminus in FIG. 2.

A “variant Fc region” comprises an amino acid sequence that differs from that of a native sequence Fc region (or fragment thereof) by virtue of at least one “amino acid substitution” as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or in the Fc region of a parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. In an alternative embodiment, a variant Fc region may be generated according to the methods herein disclosed and this variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a non-antibody polypeptide, e.g., binding domain of a receptor or ligand.

As used herein, the term “derivative” in the context of polypeptides refers to a polypeptide that comprises and amino acid sequence which has been altered by introduction of an amino acid residue substitution. The term “derivative” as used herein also refers to a polypeptide which has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide possesses a similar or identical function as the polypeptide from which it was derived. It is understood that a polypeptide comprising a variant Fc region of the present invention may be a derivative as defined herein, preferably the derivatization occurs within the Fc region.

“Substantially of equine origin” as used herein in reference to a polypeptide (e.g., an Fc region or a monoclonal antibody), indicates the polypeptide has an amino acid sequence at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94% or even more preferably at least 95%, 96%, 97%, 98% or 99% homologous to that of a native equine amino polypeptide.

The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). The preferred FcR is a native sequence FcR. Moreover, a preferred FcR is one which binds an IgG antibody Fc region, an Fc gamma receptor or “FcgR”, and includes receptors of the Fc gamma RI (FcgR1), Fc gamma RII (FcgR2), Fc gamma RIII (FcgR3) subclasses, including allelic variants and alternatively spliced forms of these receptors as well as the novel equine Fc gamma 2R. Another preferred FcR includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

The phrase “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcgRs (e.g., Natural Killer (“NK”) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cells. The primary cells for mediating ADCC in humans, NK cells, express FcgR3 only, whereas monocytes express FegR1, FcgR2 and FcgR3.

The phrases “antibody-dependent cell-mediated phagocytosis” and “ADCP” refer to a cell-mediated reaction in which phagocytic cells (e.g., macrophages, monocytes, dendritic cells) that express FcgRs (e.g., FcgR1, FcgR2a and FcgR3) recognize bound IgG antibody Fc region on a target cell and subsequently trigger a signaling cascade leading to the engulfment of the IgG-opsonized particle (e.g., bacteria, dead tissue cells).

As used herein, the phrase “effector cells” refers to leukocytes (preferably equine) which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcgR3 and perform ADCC effector function. Examples of leukocytes which mediate ADCC include PBMC, NK cells, monocytes, macrophage, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source (e.g., from blood or PBMCs). In one example, the leukocytes express FcgR1, or other relevant Fc gamma receptor, and trigger ADCP function.

A variant polypeptide with “altered” Fc receptor binding affinity is one which has either enhanced (i.e., increased, greater or higher) or diminished (i.e., reduced, decreased or lesser) Fc receptor binding affinity compared to the variant's parent polypeptide or to a polypeptide comprising a native Fc. A variant polypeptide which displays increased binding or increased binding affinity to an Fc receptor binds Fc receptor with greater affinity than the parent polypeptide. A variant polypeptide which displays decreased binding or decreased binding affinity to an Fc receptor, binds Fc receptor with lower affinity than its parent polypeptide. Such variants which display decreased binding to an Fc receptor may possess little or no appreciable binding to an Fc receptor, e.g., 0-20% binding to Fc receptor the Fc receptor compared to a parent polypeptide. A variant polypeptide which binds an Fc receptor with “enhanced affinity” as compared to its parent polypeptide, is one which binds Fc receptor with higher binding affinity than the parent polypeptide, when the amounts of variant polypeptide and parent polypeptide in a binding assay are essentially the same, and all other conditions are identical. For example, a variant polypeptide with enhanced Fc receptor binding affinity may display from about 1.10 fold to about 100 fold (more typically from about 1.2 fold to about 50 fold) increase in Fc receptor binding affinity compared to the parent polypeptide, where Fc receptor binding affinity is determined, for example, in an ELISA assay or other method available to one of ordinary skill in the art.

As used herein, an “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another different “replacement” amino acid residue. The replacement residue or residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (H is); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A “non-naturally occurring amino acid residue” refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues (s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202: 301-336 (1991).

The term “assay signal” refers to the output from any method of detecting protein-protein interactions, including but not limited to, absorbance measurements from colorimetric assays, fluorescent intensity, or disintegrations per minute. Assay formats could include ELISA, FACS, or other methods. A change in the “assay signal” may reflect a change in cell viability and/or a change in the kinetic off-rate, the kinetic on-rate, or both. A “higher assay signal” refers to the measured output number being larger than another number (e.g., a variant may have a higher (larger) measured number in an ELISA assay as compared to the parent polypeptide). A “lower” assay signal refers to the measured output number being smaller than another number (e.g., a variant may have a lower (smaller) measured number in an ELISA assay as compared to the parent polypeptide).

The term “binding affinity” refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. The binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g., seconds⁻¹) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g., molar/second). In general, it is not possible to unequivocally state whether changes in equilibrium dissociation constants (K_D or KD) are due to differences in on-rates, off-rates or both unless each of these parameters are experimentally determined (e.g., by BIACORE or SAPIDYNE measurements).

As used herein, the term “hinge region” refers to the stretch of amino acids that links the Fab antigen binding region to the Fc region of an antibody. Hinge regions of IgG subclasses may be aligned by placing the first and last cysteine residues forming inter-heavy chain disulfide (S—S) bonds in the same positions. As shown in FIG. 2, the hinge region, for example, in equine IgG constant region starts at residue 216 and extends to residue 230.

“Clq” is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. Clq together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the CDC pathway.

As used herein, the term “antibody” is used interchangeably with “immunoglobulin” or “Ig,” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or functional activity. Single chain antibodies, and chimeric, equine, or equinized antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species, are also encompassed by the present invention and the term “antibody”. The various portions of these antibodies can be joined together chemically by conventional techniques, synthetically, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or equinized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567; 4,816,397; WO 86/01533; U.S. Pat. Nos. 5,225,539; and 5,585,089 and 5,698,762. See also, Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:423-426, 1988, regarding single chain antibodies. It is understood that all forms of the antibodies comprising an Fc region (or portion thereof) are encompassed herein within the term “antibody.” Furthermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

As used herein, the term “antibody fragments” refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc′ peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. The antibody fragments preferably retain at least part of the hinge and optionally the CH1 region of an IgG heavy chain. In other preferred embodiments, the antibody fragments comprise at least a portion of the CH2 region or the entire CH2 region.

As used herein, the term “functional fragment”, when used in reference to a monoclonal antibody, is intended to refer to a portion of the monoclonal antibody that still retains a functional activity. A functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity and the like. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab′; bivalent fragments such as F(ab′)2; single chain Fv (scFv); and Fc fragments. Such terms are described in, for example, Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The term functional fragment is intended to include, for example, fragments produced by protease digestion or reduction of a monoclonal antibody and by recombinant DNA methods known to those skilled in the art.

As used herein, the term “fragment” refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more contiguous amino acid residues of the amino acid sequence of another polypeptide. In a preferred embodiment, a fragment of a polypeptide retains at least one function of the full-length polypeptide.

As used herein, the term “chimeric antibody” includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through disulfide bridges with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers associated through at least one disulfide bridge. A chimeric heavy chain of an antibody for use in equine comprises an antigen-binding region derived from the heavy chain of a non-equine antibody, which is linked to at least a portion of a equine heavy chain constant region, such as CH1 or CH2. A chimeric light chain of an antibody for use in equine comprises an antigen binding region derived from the light chain of a non-equine antibody, linked to at least a portion of a equine light chain constant region (CL). Antibodies, fragments or derivatives having chimeric heavy chains and light chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps. With this approach, hosts expressing chimeric heavy chains are separately cultured from hosts expressing chimeric light chains, and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin or fragment or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397).

As used herein, “equinized” forms of non-equine (e.g., murine) antibodies (i.e., equinized antibodies) are antibodies that contain minimal sequence, or no sequence, derived from non-equine immunoglobulin. For the most part, equinized antibodies are equine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-equine species (donor antibody) such as mouse, rat, rabbit, human or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the equine immunoglobulin are replaced by corresponding non-equine residues. Furthermore, equinized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody performance. In general, the equinized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-equine immunoglobulin and all or substantially all of the FR residues are those of a equine immunoglobulin sequence. The equinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a equine immunoglobulin.

As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding domain of a heterologous “adhesin” protein (e.g., a receptor, ligand or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e., is “heterologous”) with an immunoglobulin constant domain sequence.

As used herein, the term “ligand binding domain” refers to any native receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor. In certain embodiments, the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergene family. Other receptors, which are not members of the immunoglobulin supergene family but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectins).

As used herein, the term “receptor binding domain” refers to any native ligand for a receptor, including, e.g., cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand.

As used herein, an “isolated” polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the isolated polypeptide is purified (1) to greater than 95% by weight of polypeptides as determined by the Lowry method, and preferably, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-page under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by a least one purification step.

As used herein, the term “disorder” and “disease” are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

As used herein, the term “receptor” refers to a polypeptide capable of binding at least one ligand. The preferred receptor is a cell-surface or soluble receptor having an extracellular ligand-binding domain and, optionally, other domains (e.g., transmembrane domain, intracellular domain and/or membrane anchor). A receptor to be evaluated in an assay described herein may be an intact receptor or a fragment or derivative thereof (e.g. a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Moreover, the receptor to be evaluated for its binding properties may be present in a cell or isolated and optionally coated on an assay plate or some other solid phase or labeled directly and used as a probe.

As used herein a variant polypeptide that knocks out, or knocks down, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) in the presence of equine effector cells compared to parent antibody is one which in vitro or in vivo is substantially less active at mediating ADCC, ADCP and/or CDC, when the amounts of variant polypeptide and parent antibody used in the assay are essentially the same. For example, such a variant causes a lower, preferably negligible, amount of target cell lysis or phagocytosis in a given ADCC, ADCP or CDC assay than the parent polypeptide in an identical ADCC assay. Such variants may be identified, for example, using an ADCC, ADCP or CDC assay, but other assays or methods for determining ADCC, ADCP or CDC activity may also be employed (e.g., animal models). In preferred embodiments, the variant polypeptide is about 100, 75, 50, or 25 percent less active at mediating ADCC, ADCP and CDC than the parent polypeptide.

Equine Wildtype IgG

Equine IgGs are well known in the art and fully described in, for example, Wagner et al., 2004, J. Immunol., vol. 173, pages 3230-3242; Wagner, 2006, Dev. Comp. Immunol., vol. 30, pages 155-164; Sheoran et al., 2000, Am. J. Vet. Res., vol 61, pages 1099-1105; and Wagner et al., 1998, Immunobiology, vol. 199 (1), pages 105-118.

In one embodiment, equine IgG is IgG1. In another embodiment, equine IgG is IgG2. In another embodiment, equine IgG is IgG3. In another embodiment, equine IgG is IgG4. In another embodiment, equine IgG is IgG5. In another embodiment, equine IgG is IgG6. In another embodiment, equine IgG is IgG7. In a particular example, equine IgG is IgG1.

The amino acid and nucleic acid sequences of IgGs1-7 are also well known in the art.

In one example, IgG of the invention comprises a constant domain, for example, CH1, CH2, or CH3 domains, or a combination thereof. In another example, the constant domain of the invention comprises Fc region, including, for example, CH2 or CH3 domains or a combination thereof.

In a particular example, the wild-type constant domain comprises any one of the amino acid sequences set forth in SEQ ID NOs.: 1-7. In a particular embodiment, the wild-type constant domain of IgG1, 2, 3, 4, 5, 6, and 7 comprises the amino acid sequence set forth in SEQ ID NO.: 1, 2, 3, 4, 5, 6, and 7, respectively. In some embodiments, the wild-type IgG constant domain is a homologue, a variant, an isomer, or a functional fragment of any one of SEQ ID NOs.: 1-7, but without any mutation described herein. Each possibility represents a separate embodiment of the present invention. For example, in one embodiment, in a particular embodiment, the wild-type constant domain of IgG1 comprises the amino acid sequence set forth in SEQ ID NO.: 1.

IgG constant domains also include polypeptides with amino acid sequences substantially similar to the amino acid sequence of the heavy and/or light chain. Substantially the same amino acid sequence is defined herein as a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).

The present invention also includes nucleic acid molecules that encode IgGs or portion thereof, described herein. In one embodiment, the nucleic acids may encode an antibody heavy chain comprising, for example, CH1, CH2, CH3 regions, or a combination thereof. In another embodiment, the nucleic acids may encode an antibody heavy chain comprising, for example, any one of the VH regions or a portion thereof, or any one of the VH CDRs, including any variants thereof. The invention also includes nucleic acid molecules that encode an antibody light chain comprising, for example, any one of the CL regions or a portion thereof, any one of the VL regions or a portion thereof or any one of the VL CDRs, including any variants thereof. In certain embodiments, the nucleic acid encodes both a heavy and light chain, or portions thereof.

The amino acid sequence of the wild-type constant domain set forth in SEQ ID NO.: 1, 2, 3, 4, 5, 6, or 7 is encoded by its corresponding nucleic acid sequence.

Modified Equine IgG

The inventors of the instant application have found that substituting the amino acid residue at position 252, 286, 311, 312, 426, 428, 434, or 436 with another amino acid surprisingly and unexpectedly exhibited a desired effect. The term, position, as used herein, refers to a position numbered according to the Eu index as in Kabat (Kabat el al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In one embodiment, the desired effect is a higher affinity to FcRn, relative to an IgG having the wild-type equine IgG constant domain. In another embodiment, the desired effect is eliminating or reducing complement-dependent cytotoxicity, relative to an IgG having the wild-type equine IgG constant domain. In another embodiment, the desired effect is eliminating or reducing antibody-dependent cellular phagocytosis, relative to an IgG having the wild-type equine IgG constant domain. In yet another embodiment, the desired effect is eliminating or reducing the binding of the IgG to Fc gamma receptor (eFcgR).

In one embodiment, the invention provides a modified IgG comprising: a equine IgG constant domain comprising at least one amino acid substitution relative to a wild-type equine IgG constant domain, wherein the substitution is at amino acid residue 252, 286, 311, 312, 426, 428, 434, or 436, numbered according to the Eu index as in Kabat. The amino acid at these positions can be substituted with any other amino acid. Examples of substitution amino acid includes, for example, but not limited to, asparagine, histidine, serine, alanine, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, and proline. In some embodiments, the substitution amino acid is a non-natural amino acid.

The modified equine IgG of the invention can be any suitable equine IgG, known to one of skilled in the art. Examples of the modified equine IgG include a modified variant of IgG1, 2, 3, 4, 5, 6, or 7.

In an exemplary embodiment, the modified equine IgG is a modified equine IgG1.

In another exemplary embodiment, the equine IgG1 constant domain comprises one or more of substitution mutations M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, G426A, G426C, G426D, G426E, G426F, G426H, G426I, G426K, G426L, G426M, G426N, G426P, G426Q, G426R, G426S, G426T, G426V, G426W, G426Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W.

In another example, the mutant IgG1 constant domain of the invention comprises one or more mutations described herein. In some embodiments, the mutant IgG1 constant domain is a homologue, a variant, an isomer, or a functional fragment, but with mutation of the invention described herein. Each possibility represents a separate embodiment of the present invention.

The amino acid sequence of the mutant constant domain is encoded by its corresponding mutant nucleic acid sequence.

Methods for Making Antibody Molecules of the Invention

Methods for making antibody molecules are well known in the art and fully described in U.S. Pat. Nos. 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023 and U.S. Patent Application Publication 20060067930, which are incorporated by reference herein in their entirety. Any suitable method, process, or technique, known to one of skilled in the art, can be used. An antibody molecule having a variant Fc region of the invention may be generated according to the methods well known in the art. In some embodiments, the variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or binding domain of a receptor or ligand.

With the advent of methods of molecular biology and recombinant technology, a person of skilled in the art can produce antibody and antibody-like molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies. Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with assembly of the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources.

Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, large cell cultures of laboratory or commercial size, using transgenic plants, or by direct chemical synthesis employing no living organisms at any stage of the process, all antibodies have a similar overall 3 dimensional structure. This structure is often given as H2L2 and refers to the fact that antibodies commonly comprise two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as “variable” or ‘V” regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity. The variable regions of either H or L chains contain the amino acid sequences capable of specifically binding to antigenic targets.

As used herein, the term “antigen binding region” refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody binding region includes the “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the H or L chains that provide for the antigen binding regions are smaller sequences dubbed “hypervariable” because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as “complementarity determining regions” or “CDR” regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called “framework regions” made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

The present invention further provides a vector including at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an antibody or portion. Such “codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using the “codon usage rules” of Lathe, a single nucleotide sequence, or a set of nucleotide sequences that contains a theoretical “most probable” nucleotide sequence capable of encoding equine IgG sequences can be identified. It is also intended that the antibody coding regions for use in the present invention could also be provided by altering existing antibody genes using standard molecular biological techniques that result in variants of the antibodies and peptides described herein. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the antibodies or peptides.

For example, one class of substitutions is conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a equine antibody peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Iie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg, replacements among the aromatic residues Phe, Tyr, and the like. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990).

Variant equine antibodies or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as epitope binding or in vitro ADCC activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as epitope mapping (e.g., HDX), crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

Moreover, polypeptides often contain amino acids other than the twenty “naturally occurring” amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP ribosylation, for instance, are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., T. E. Creighton, W. H. Freeman & Co., N.Y., 1993). Many detailed reviews are available on this subject, such as by Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, N.Y., 1983); Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

In another aspect, the invention provides antibody derivatives. A “derivative” of an antibody contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well-known in the art, is useful for cross-linking the antibody or fragment to a water-insoluble support matrix or to other macromolecular carriers.

Derivatives also include radioactively labeled monoclonal antibodies that are labeled. For example, with radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium (H³) or the like; conjugates of monoclonal antibodies with biotin or avidin, with enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemoluminescent agents (such as acridine esters) or fluorescent agents (such as phycobiliproteins).

Another derivative bifunctional antibody of the invention is a bispecific antibody, generated by combining parts of two separate antibodies that recognize two different antigenic groups. This may be achieved by crosslinking or recombinant techniques. Additionally, moieties may be added to the antibody or a portion thereof to increase half-life in vivo (e.g., by lengthening the time to clearance from the blood stream. Such techniques include, for example, adding PEG moieties (also termed pegylation), and are well-known in the art. See U.S. Patent. Appl. Pub. No. 20030031671.

In some embodiments, the nucleic acids encoding a subject antibody are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37° C., sometimes under selection, for a period of about 1-24 hours in order to allow for the expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the media in which the cell is growing. Traditionally, monoclonal antibodies have been produced as native molecules in murine hybridoma lines. In addition to that technology, the present invention provides for recombinant DNA expression of the antibodies. This allows the production of antibodies, as well as a spectrum of antibody derivatives and fusion proteins in a host species of choice.

A nucleic acid sequence encoding at least one antibody, portion or polypeptide of the invention may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 supra, may be used to construct nucleic acid sequences which encode an antibody molecule or antigen binding region thereof.

A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as peptides or antibody portions in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook et al., 2001 supra; Ausubel et al., 1993 supra.

The present invention accordingly encompasses the expression of an antibody or peptide, in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin. Any other suitable mammalian cell, known in the art, may also be used.

In one embodiment, the nucleotide sequence of the invention will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. See, e.g., Ausubel et al., 1993 supra. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, CoIE1, pSC101, pACYC 184, .pi.vX). Such plasmids are, for example, disclosed by Maniatis et al., 1989 supra; Ausubel et al, 1993 supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include p1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987), and Streptomyces bacteriophages such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986); lzaki, 33 Jpn. J. Bacteriol. 729-42 (1978); and Ausubel et al., 1993 supra.

Alternatively, gene expression elements useful for the expression of cDNA encoding antibodies or peptides include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40 (Okayama et al., 1983).

Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part genomic DNA (Whittle et al., 1 Protein Engin. 499 (1987)), the transcriptional promoter can be human cytomegalovirus, the promoter enhancers can be cytomegalovirus and mouse/human immunoglobulin, and mRNA splicing and polyadenylation regions can be the native chromosomal immunoglobulin sequences.

In one embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells.

Each fused gene can be assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene product are then transfected singly with a peptide or H or L chain-encoding gene, or are co-transfected with H and L chain gene. The transfected recipient cells are cultured under conditions that permit expression of the incorporated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

In one embodiment, the fused genes encoding the peptide or H and L chains, or portions thereof are assembled in separate expression vectors that are then used to cotransfect a recipient cell. Alternatively the fused genes encoding the H and L chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibody, the recipient cell line may be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as B lymphocytes of equine or non-equine origin, hybridoma cells of equine or non-equine origin, or interspecies heterohybridoma cells.

The expression vector carrying an antibody construct or polypeptide of the invention can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

Yeast may provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of peptides, antibodies, fragments and regions thereof. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches can be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) TRL Press, Oxford, UK 1985).

Bacterial strains can also be utilized as hosts for the production of antibody molecules or peptides described by this invention. Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches can be taken for evaluating the expression plasmids for the production of antibodies, fragments and regions or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, N.Y. (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001).

Host mammalian cells may be grown in vitro or in vivo. Mammalian cells provide posttranslational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells which can be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems are available for the expression of cloned peptides Hand L chain genes in mammalian cells (see Glover, 1985 supra). Different approaches can be followed to obtain complete H2L2 antibodies. It is possible to co-express Hand L chains in the same cells to achieve intracellular association and linkage of Hand L chains into complete tetrameric H2L2 antibodies and/or peptides. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both Hand L chains and/or peptides can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. cell lines producing peptides and/or H2L2 molecules via either route could be transfected with plasmids encoding additional copies of peptides, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines.

For long-term, high-yield production of recombinant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluation of compounds/components that interact directly or indirectly with the antibody molecule.

Once an antibody of the invention has been produced, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In many embodiments, antibodies are secreted from the cell into culture medium and harvested from the culture medium.

Pharmaceutical and Veterinary Applications

The invention also provides a pharmaceutical composition comprising molecules of the invention and one or more pharmaceutically acceptable carriers. More specifically, the invention provides for a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or peptide according to the invention.

“Pharmaceutically acceptable carriers” include any excipient which is nontoxic to the cell or animal being exposed thereto at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents.

“Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents and absorption delaying agents.

Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; as well as combinations thereof.

The pharmaceutical compositions of the invention may be formulated in a variety of ways, including for example, liquid, semi-solid, or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the compositions are in the form of injectable or infusible solutions. The composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The composition may be formulated as an immediate, controlled, extended or delayed release composition.

The compositions of the invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibodies (typically carried in a pharmaceutical formulation) to an airway surface. Topical administration to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler). Topical administration of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.

In some desired embodiments, the antibodies are administered by parenteral injection. For parenteral administration, antibodies or molecules can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier such vehicles are water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine and the like. Liposomes and nonaqueous vehicles such as fixed oils can also be used. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjustment agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15% or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

The antibodies or molecules of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins. Any suitable lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate. The compositions containing the present antibodies or a cocktail thereof can be administered for prevention of recurrence and/or therapeutic treatments for existing disease. Suitable pharmaceutical carriers are described in the most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference text in this field of art. In therapeutic application, compositions are administered to a subject already suffering from a disease, in an amount sufficient to cure or at least partially arrest or alleviate the disease and its complications.

Effective doses of the compositions of the present invention, for treatment of conditions or diseases as described herein vary depending upon many different factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; target site; physiological state of the animal; other medications administered; whether treatment is prophylactic or therapeutic; age, health, and weight of the recipient; nature and extent of symptoms kind of concurrent treatment, frequency of treatment, and the effect desired.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating veterinarian. In any event, the pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the subject.

Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

The pharmaceutical compositions of the invention may include a “therapeutically effective amount.” A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

In another aspect, the compositions of the invention can be used, for example, in the treatment of various diseases and disorders in equine. As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.

All patents and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention.

EXAMPLES

Example 1

Generating Equine IgG and FcRn

NCBI database sequence AJ300675.1 (Wagner et al., 1998) was used to generate all equine IgG1-based mAbs reported herein.

The equine FcRn (large alpha subunit p51) protein (NCBI: XP_023505910.1) was generated recombinantly. The equine beta-2-microglobulin (B2M) small subunit (NCBI: NM_001082502.2) that associates with FcRn to form a functional complex was also generated recombinantly and used in surface plasmon resonance (SPR) binding affinity experiments.

DNA for equine FcRn/B2M and all equine mAb genes were codon-optimized for mammalian expression, and constructs were transiently expressed either in HEK 293 cells using a standard lipofectamine transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or into CHO cells using the ExpiCHO transient system (ThermoFisher Scientific) kit protocols. ExpiCHO expression followed protocols outlined by ThermoFisher for either mAb or FcRn/B2M transfection. For mAbs, plasmid containing gene sequence encoding for an IgG kappa light chain was co-transfected with a plasmid encoding for IgG heavy chain. For HEK293 expression, equal amounts by weight of heavy chain plasmid and kappa chain plasmid were co-transfected. For FcRn/B2M, the two plasmids encoding each were transfected. Cells were allowed to grow for 7 days (HEK293) or 12 days (CHO) after which supernatants were collected for protein purification. mAbs were screened for binding to protein A or protein G sensors via Octet QKe quantitation (Pall ForteBio Corp, Menlo Park, CA, USA). Expression was quantified on Octet with protein A or protein G sensors using standard curves, and mAbs were purified with protein G or protein A/G affinity chromatography. For all protein constructs, Sodium Acetate pH 5.5 was used as binding and wash buffer, and elution was performed at pH 3.4. The purified proteins were neutralized and dialyzed into 20 mM Na acetate, pH 5.5, 140 mM NaCl for further analysis. The FcRn plasmid contained a c-terminal His tag, thus the FcRn/B2M complex was purified by IMAC affinity purification. The concentration of the mAbs and FcRn/B2M proteins was measured via NanoDrop at 280 nm. Protein quality was assessed via analytical SEC and standard coomassie protein gels.

The purified FcRn/B2M was biotinylated as follows. The purified FcRn/B2M protein was dialyzed into 10 mM Tris-HCl, pH 8.0 and concentrated using AmiconUltra,10KMWCO (EMD Millipore, Billerica, MA). The Biotin Acceptor Peptide (BAP) AGLNDIFEAQKIEWHE which was expressed at the c-terminus of the receptor allowed for transfer of biotin to this stretch of amino acids using the biotin ligase BirA. Biotinylation reactions were carried out as described in the manufacturer protocol (Avidity, LLC, Aurora, CO). The FcRn/B2M receptor was then dialyzed into PBS to remove residual biotin.

Example 2

Construction of Equine IgG Fc Mutants

Plasmids containing sequence encoding for equine constant regions for the IgG1 were utilized and VH/VL sequences for each mAb investigated herein were inserted upstream and in frame with the nucleotides encoding for the constant domains. Mutations were incorporated into either CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant region as gene fragment and were subsequently sub-cloned into respective variable region of interest.

Expression and Purification

The monoclonal antibody (mAbs) mutants were expressed in mammalian suspension cell systems, EXPICHO-S(Chinese Hamster Ovary) cells, obtained from Thermo Fisher. Suspension EXPICHO-S cells were maintained in EXPICHO expression medium (Gibco) between 0.14 and 8.0×10e6 cells/ml. Cells were diluted following the ExpiCHO Protocol user manual on Day −1 and transfection day. Diluted cells were transfected as described in the protocol using reagents sourced from ExpiFectamine CHO Transfection Kit (Gibco) following Max Titer conditions. Following 12-14 days of incubation, the cultures were harvested and clarified. Antibodies were purified from the clarified supernatant via Protein A chromatography over MabSelect Sure LX (GE Healthcare) which had been pre-equilibrated with PBS. Following sample load, the resin was washed with PBS and then with 20 mM sodium acetate, pH 5.5. Samples were eluted from the column with 20 mM acetic acid, pH 3.5. Following elution, pools were made and neutralized with the addition of 1 M sodium acetate to 4%. Depending on available volume and intended use, samples were sometimes exchanged into a final buffer (e.g. PBS, other). Concentration was measured by absorbance at 280 nm.

SDS-PAGE

Non-reduced (nr) and reduced sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) was performed using 4-12% Bis-Tris NuPAGE gels in MES-SDS running buffer, and SeeBlue Plus 2 standards, all from Invitrogen. For non-reduced samples, 1 mM of alkylating agent N-ethylmaleimide (NEM) was added, for reduced samples reducing agent dithiothreitol (DTT) was added. Gels were stained with Coomassie Blue to detect the protein bands.

Example 3

FcRn Binding Assay

Biacore Method for eFcRn:

Equine Fc-based antibodies or fusion protein binding affinities to equine FcRn were determined by surface plasmon resonance (SPR). All reported KD's were measured in Biacore T200 (Cytiva, Marlborough, MA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA) using SA sensor. Equine FcRn was captured on the surface of the sensor for a desired surface density. Running buffer of 20 mM MES, 150 mM NaCl, 0.005% Tween 20, 0.5 mg/mL BSA, pH 6 and/or PBS, 0.0005% Tween 20, pH7.4 were used. Various concentrations of equine mAbs were titrated in proper running buffer and flowed over the receptor surface. Regeneration was performed with 50 mM Tris-HCl, pH8. Kinetic binding affinity was analyzed using Biacore T200 Evaluation software (Cytiva, Marlborough, MA, USA) or Biacore 8K Insight Evaluation Software with method of double referencing: the reference flow cell was subtracted from the flow cell containing immobilized equine FcRn and blank runs containing buffer only were subtracted out from all runs. The resulting curve was fitted with the 1:1 binding model. Runs were performed at 25° C.

Mutations made at respective positions have a marked effect on the affinity of the IgG to FcRn at pH6.

Binding of wild-type (WTs) and mutant IgG1, 4, and 7 to equine FcRn were measured by surface plasmon resonance (Biacore). The results of the binding affinity of mutant IgG1, 4, and 7 to equine FcRn are shown in Tables 1, 2, and 3, respectively, below.

TABLE 1
Effect of IgG1 mutants on eFcRn binding affinity.

	Mutations as	Equine IgG1 + FcRn binding
	per EU	affinity

ID	numbering	KD at	KD at	Codon
No.	system	pH 6	pH 7.4	Usage

1	WT	2.25E−08	LS
2	M252A	5.03E−08	LS	GCC
3	M252C	LS	LS	TGC
4	M252D	LS	LS	GAC
5	M252E	LS	LS	GAG
6	M252F	4.75E−08	LS	TTC
7	M252G	LS	LS	GGC
8	M252H	LS	LS	CAC
9	M252I	LS	LS	ATC
10	M252K	3.67E−08	LS	AAG
11	M252L	1.01E−07	LS	CTG
12	M252N	LS	LS	AAC
13	M252P	LS	LS	CCC
14	M252Q	LS	LS	CAG
15	M252R	LS	LS	AGG
16	M252S	LS	LS	TCC
17	M252T	LS	LS	ACC
18	M252V	LS	LS	GTG
19	M252W	2.66E−08	LS	TGG
20	M252Y	1.93E−08	LS	TAC
21	T286A	1.85E−08	LS	GCC
22	T286C	3.54E−08	LS	TGC
23	T286D	2.39E−08	LS	GAC
24	T286E	2.19E−08	LS	GAG
25	T286F	1.85E−08	LS	TTC
26	T286G	1.50E−08	LS	GGC
27	T286H	2.24E−08	LS	CAC
28	T286I	1.82E−08	LS	ATC
29	T286K	3.73E−08	LS	AAG
30	T286L	1.23E−08	LS	CTG
31	T286M	1.56E−08	LS	ATG
32	T286N	LS	LS	AAC
33	T286P	1.46E−08	LS	CCC
34	T286Q	1.70E−08	LS	CAG
35	T286R	2.05E−08	LS	AGG
36	T286S	1.31E−08	LS	TCC
37	T286V	1.62E−08	LS	GTG
38	T286W	1.84E−08	LS	TGG
39	T286Y	1.50E−08	LS	TAC
40	Q311A	2.10E−08	LS	GCC
41	Q311C	1.57E−08	LS	TGC
42	Q311D	5.46E−08	LS	GAC
43	Q311E	6.73E−08	LS	GAG
44	Q311F	2.03E−08	LS	TTC
45	Q311G	2.01E−08	LS	GGC
46	Q311H	2.43E−08	LS	CAC
47	Q311I	1.26E−08	LS	ATC
48	Q311K	1.47E−08	LS	AAG
49	Q311L	1.61E−08	LS	CTG
50	Q311M	1.20E−08	LS	ATG
51	Q311N	2.43E−08	LS	AAC
52	Q311P	LS	LS	CCC
53	Q311R	1.01E−08	LS	AGG
54	Q311S	2.44E−08	LS	TCC
55	Q311T	1.74E−08	LS	ACC
56	Q311V	1.63E−08	LS	GTG
57	Q311W	2.12E−08	LS	TGG
58	Q311Y	2.12E−08	LS	TAC
59	D312A	9.86E−09	LS	GCC
60	D312C	2.10E−08	LS	TGC
61	D312E	2.52E−08	LS	GAG
62	D312F	1.78E−08	LS	TTC
63	D312G	1.62E−08	LS	GGC
64	D312H	1.71E−08	LS	CAC
65	D312I	LS	LS	ATC
66	D312K	1.58E−08	LS	AAG
67	D312L	2.46E−08	LS	CTG
68	D312M	2.34E−08	LS	ATG
69	D312N	2.23E−08	LS	AAC
70	D312P	8.88E−09	LS	CCC
71	D312Q	4.60E−08	LS	CAG
72	D312R	LS	LS	AGG
73	D312S	1.67E−08	LS	TCC
74	D312T	2.03E−08	LS	ACC
75	D312V	8.32E−09	LS	GTG
76	D312W	2.16E−08	LS	TGG
77	D312Y	1.71E−08	LS	TAC
78	G426A	1.76E−08	LS	GCC
79	G426C	2.04E−08	LS	TGC
80	G426D	2.53E−08	LS	GAC
81	G426E	2.41E−08	LS	GAG
82	G426F	1.31E−08	LS	TTC
83	G426H	1.28E−08	LS	CAC
84	G426I	9.47E−09	LS	ATC
85	G426K	9.30E−09	LS	AAG
86	G426L	9.61E−09	LS	CTG
87	G426M	9.09E−09	LS	ATG
88	G426N	1.03E−08	LS	AAC
89	G426P	2.39E−08	LS	CCC
90	G426Q	9.01E−09	LS	CAG
91	G426R	1.03E−08	LS	AGG
92	G426S	8.97E−09	LS	TCC
93	G426T	9.73E−09	LS	ACC
94	G426V	8.79E−09	LS	GTG
95	G426W	1.47E−08	LS	TGG
96	G426Y	1.19E−08	LS	TAC
97	M428A	7.60E−08	LS	GCC
98	M428C	4.75E−08	LS	TGC
99	M428D	LS	LS	GAC
100	M428E	4.39E−08	LS	GAG
101	M428F	1.73E−08	LS	TTC
102	M428G	4.88E−08	LS	GGC
103	M428H	3.31E−08	LS	CAC
104	M428I	2.80E−08	LS	ATC
105	M428K	2.87E−08	LS	AAG
106	M428L	8.76E−09	LS	CTG
107	M428N	5.16E−08	LS	AAC
108	M428P	LS	LS	CCC
109	M428Q	3.29E−08	LS	CAG
110	M428R	LS	LS	AGG
111	M428S	LS	LS	TCC
112	M428T	3.48E−08	LS	ACC
113	M428V	6.58E−08	LS	GTG
114	M428W	3.19E−08	LS	TGG
115	M428Y	1.70E−08	LS	TAC
116	N434A	6.54E−09	LS	GCC
117	N434C	4.26E−08	LS	TGC
118	N434D	LS	LS	GAC
119	N434E	5.06E−08	LS	GAG
120	N434F	2.83E−09	1.46E−05	TTC
121	N434G	5.57E−09	LS	GGC
122	N434H	4.10E−09	LS	CAC
123	N434I	4.43E−08	LS	ATC
124	N434K	1.75E−08	LS	AAG
125	N434L	LS	LS	CTG
126	N434M	2.90E−08	LS	ATG
127	N434P	2.50E−08	LS	CCC
128	N434Q	1.28E−08	LS	CAG
129	N434R	1.40E−08	LS	AGG
130	N434S	1.00E−08	LS	TCC
131	N434T	1.96E−08	LS	ACC
132	N434V	3.12E−08	LS	GTG
133	N434W	1.86E−09	7.31E−06	TGG
134	N434Y	2.15E−09	LS	TAC
135	Y436A	2.04E−08	LS	GCC
136	Y436C	3.28E−08	LS	TGC
137	Y436D	2.84E−08	LS	GAC
138	Y436E	3.38E−08	LS	GAG
139	Y436F	1.25E−08	LS	TTC
140	Y436G	2.20E−08	LS	GGC
141	Y436H	1.91E−08	LS	CAC
142	Y436I	7.84E−09	LS	ATC
143	Y436K	1.80E−08	LS	AAG
144	Y436L	1.24E−08	LS	CTG
145	Y436M	1.11E−08	LS	ATG
146	Y436N	2.31E−08	LS	AAC
147	Y436P	LS	LS	CCC
148	Y436Q	1.46E−08	LS	CAG
149	Y436R	2.56E−08	LS	AGG
150	Y436S	1.35E−08	LS	TCC
151	Y436T	1.52E−08	LS	ACC
152	Y436V	9.06E−09	LS	GTG
153	Y436W	1.02E−08	LS	TGG
Mutants are numbered according to the EU Index as in Kabat.
LS = Low Signal.

TABLE 2
Effect of IgG4 mutants on eFcRn binding affinity.

	Mutations as	Equine IgG4 + FcRn binding
	per EU	affinity

ID	numbering	KD at	KD at	Codon
No.	system	pH 6	pH 7.4	Usage

1	G4_WT	22.5E−9	NBO	—
2	K311A	24.8E−9	NBO	GCC
3	K311C	23.5E−9	NBO	TGC
4	K311D	64.3E−9	NBO	GAC
5	K311E	75.2E−9	NBO	GAG
6	K311F	41.8E−9	NBO	TTC
7	K311G	51.2E−9	NBO	GGC
8	K311H	35.6E−9	NBO	CAC
9	K311I	30.6E−9	NBO	ATC
10	K311L	28.0E−9	NBO	CTG
11	K311M	16.4E−9	NBO	ATG
12	K311N	41.1E−9	NBO	AAC
13	K311P	2.0E−6	NBO	CCC
14	K311Q	21.9E−9	NBO	CAG
15	K311R	16.1E−9	NBO	AGG
16	K311S	12.4E−9	NBO	TCC
17	K311T	25.5E−9	NBO	ACC
18	K311V	22.4E−9	NBO	GTG
19	K311W	27.1E−9	NBO	TGG
20	K311Y	21.3E−9	NBO	TAC
21	A426C	18.2E−9	NBO	TGC
22	A426D	15.5E−9	NBO	GAC
23	A426E	18.2E−9	NBO	GAG
24	A426F	17.6E−9	NBO	TTC
25	A426G	33.3E−9	NBO	GGC
26	A426H	17.8E−9	NBO	CAC
27	A426I	17.4E−9	NBO	ATC
28	A426K	14.5E−9	NBO	AAG
29	A426L	17.4E−9	NBO	CTG
30	A426M	18.5E−9	NBO	ATG
31	A426N	18.7E−9	NBO	AAC
32	A426P	9.8E−9	NBO	CCC
33	A426Q	21.2E−9	NBO	CAG
34	A426R	22.0E−9	NBO	AGG
35	A426S	21.1E−9	NBO	TCC
36	A426T	15.8E−9	NBO	ACC
37	A426V	18.8E−9	NBO	GTG
38	A426W	14.4E−9	NBO	TGG
39	A426Y	12.3E−9	NBO	TAC
40	M428A	23.0E−6	NBO	GCC
41	M428C	53.2E−9	NBO	TGC
42	M428D	40.7E−9	NBO	GAC
43	M428E	40.8E−9	NBO	GAG
44	M428F	25.4E−9	NBO	TTC
45	M428G	31.5E−9	NBO	GGC
46	M428H	29.4E−9	NBO	CAC
47	M428I	49.5E−9	NBO	ATC
48	M428K	46.2E−9	NBO	AAG
49	M428L	17.4E−9	NBO	CTG
50	M428N	44.3E−9	NBO	AAC
51	M428P	13.0E−6	NBO	CCC
52	M428Q	30.0E−9	NBO	CAG
53	M428R	93.1E−9	NBO	AGG
54	M428S	40.6E−9	NBO	TCC
55	M428T	71.8E−9	NBO	ACC
56	M428V	75.2E−9	NBO	GTG
57	M428W	31.0E−9	NBO	TGG
58	M428Y	30.8E−9	NBO	TAC
59	N434A	17.4E−9	NBO	GCC
60	N434C	112.8E−9	NBO	TGC
61	N434D	38.5E−9	NBO	GAC
62	N434E	71.3E−9	NBO	GAG
63	N434F	4.4E−9	NBO	TTC
64	N434G	21.2E−9	NBO	GGC
65	N434H	5.2E−9	NBO	CAC
66	N434I	40.6E−9	NBO	ATC
67	N434K	42.8E−9	NBO	AAG
68	N434L	48.5E−9	NBO	CTG
69	N434M	43.4E−9	NBO	ATG
70	N434P	39.4E−9	NBO	CCC
71	N434Q	17.7E−9	NBO	CAG
72	N434R	22.7E−9	NBO	AGG
73	N434S	23.8E−9	NBO	TCC
74	N434T	57.5E−9	NBO	ACC
75	N434V	32.6E−9	NBO	GTG
76	N434W	1.6E−9	NBO	TGG
77	N434Y	4.6E−9	NBO	TAC
78	D312A	13.1E−9	NBO	GCC
79	D312C	27.1E−9	NBO	TGC
80	D312E	50.2E−9	NBO	GAG
81	D312F	23.0E−9	NBO	TTC
82	D312G	11.2E−9	NBO	GGC
83	D312H	20.2E−9	NBO	CAC
84	D312I	29.4E−9	NBO	ATC
85	D312K	13.6E−9	NBO	AAG
86	D312L	11.6E−9	NBO	CTG
87	D312M	25.0E−9	NBO	ATG
88	D312N	16.1E−9	NBO	AAC
89	D312P	15.1E−9	NBO	CCC
90	D312Q	28.1E−9	NBO	CAG
91	D312R	18.5E−9	NBO	AGG
92	D312S	17.4E−9	NBO	TCC
93	D312T	28.7E−9	NBO	ACC
94	D312V	31.1E−9	NBO	GTG
95	D312W	12.1E−9	NBO	TGG
96	D312Y	19.1E−9	NBO	TAC
97	Y436A	21.6E−9	NBO	GCC
98	Y436C	57.0E−9	NBO	TGC
99	Y436D	25.5E−9	NBO	GAC
100	Y436E	91.5E−9	NBO	GAG
101	Y436F	12.6E−9	NBO	TTC
102	Y436G	34.1E−9	NBO	GGC
103	Y436H	61.6E−9	NBO	CAC
104	Y436I	12.0E−9	NBO	ATC
105	Y436K	20.3E−9	NBO	AAG
106	Y436L	16.0E−9	NBO	CTG
107	Y436M	14.3E−9	NBO	ATG
108	Y436N	49.8E−9	NBO	AAC
109	Y436P	NBO	NBO	CCC
110	Y436Q	25.6E−9	NBO	CAG
111	Y436R	48.3E−9	NBO	AGG
112	Y436S	30.9E−9	NBO	TCC
113	Y436T	27.2E−9	NBO	ACC
114	Y436V	13.2E−9	NBO	GTG
115	Y436W	20.2E−9	NBO	TGG
116	T286A	48.7E−9	NBO	GCC
117	T286C	34.4E−9	NBO	TGC
118	T286D	492.2E−9	NBO	GAC
119	T286E	251.8E−9	NBO	GAG
120	T286F	22.2E−9	NBO	TTC
121	T286G	23.3E−9	NBO	GGC
122	T286H	27.1E−9	NBO	CAC
123	T286I	13.3E−9	NBO	ATC
124	T286K	24.7E−9	NBO	AAG
125	T286L	17.5E−9	NBO	CTG
126	T286M	20.3E−9	NBO	ATG
127	T286N	NBO	NBO	AAC
128	T286P	5.2E−9	NBO	CCC
129	T286Q	24.5E−9	NBO	CAG
130	T286R	21.4E−9	NBO	AGG
131	T286S	24.1E−9	NBO	TCC
132	T286V	15.3E−9	NBO	GTG
133	T286W	45.0E−9	NBO	TGG
134	T286Y	26.9E−9	NBO	TAC
Mutants are numbered according to the EU Index as in Kabat.
NBO = No Binding Observed.

TABLE 3
Effect of IgG7 mutants on eFcRn binding affinity.

	Mutations as	Equine IgG7 + FcRn binding
	per EU	affinity

ID	numbering	KD at	KD at	Codon
No.	system	pH 6	pH 7.4	Usage

1	G7_WT	21.7E−9	NBO	—
2	K311A	24.4E−9	NBO	GCC
3	K311C	15.9E−9	NBO	TGC
4	K311D	34.1E−9	NBO	GAC
5	K311E	60.0E−9	NBO	GAG
6	K311F	20.5E−9	NBO	TTC
7	K311G	20.9E−9	NBO	GGC
8	K311H	19.0E−9	NBO	CAC
9	K311I	15.1E−9	NBO	ATC
10	K311L	17.3E−9	NBO	CTG
11	K311M	10.4E−9	NBO	ATG
12	K311N	38.0E−9	NBO	AAC
13	K311P	LS	NBO	CCC
14	K311Q	39.5E−9	NBO	CAG
15	K311R	11.8E−9	NBO	AGG
16	K311S	27.6E−9	NBO	TCC
17	K311T	19.4E−9	NBO	ACC
18	K311V	21.3E−9	NBO	GTG
19	K311W	22.1E−9	NBO	TGG
20	K311Y	18.9E−9	NBO	TAC
21	A426C	14.4E−9	NBO	TGC
22	A426D	18.9E−9	NBO	GAC
23	A426E	13.4E−9	NBO	GAG
24	A426F	13.2E−9	NBO	TTC
25	A426G	13.6E−9	NBO	GGC
26	A426H	18.4E−9	NBO	CAC
27	A426I	12.6E−9	NBO	ATC
28	A426K	17.8E−9	NBO	AAG
29	A426L	15.6E−9	NBO	CTG
30	A426M	22.7E−9	NBO	ATG
31	A426N	15.8E−9	NBO	AAC
32	A426P	23.2E−9	NBO	CCC
33	A426Q	18.6E−9	NBO	CAG
34	A426R	35.0E−9	NBO	AGG
35	A426S	40.4E−9	NBO	TCC
36	A426T	26.5E−9	NBO	ACC
37	A426V	23.3E−9	NBO	GTG
38	A426W	24.1E−9	NBO	TGG
39	A426Y	21.1E−9	NBO	TAC
40	M428A	LS	NBO	GCC
41	M428C	14.6E−6	NBO	TGC
42	M428D	2.0E−6	NBO	GAC
43	M428E	39.8E−9	NBO	GAG
44	M428F	23.9E−9	NBO	TTC
45	M428G	96.9E−9	NBO	GGC
46	M428H	165.0E−9	NBO	CAC
47	M428I	240.0E−9	NBO	ATC
48	M428K	63.5E−9	NBO	AAG
49	M428L	10.5E−9	NBO	CTG
50	M428N	NBO	NBO	AAC
51	M428P	NBO	NBO	CCC
52	M428Q	20.0E−9	NBO	CAG
53	M428R	1.2E−9	NBO	AGG
54	M428S	5.8E−9	NBO	TCC
55	M428T	NBO	NBO	ACC
56	M428V	NBO	NBO	GTG
57	M428W	6.6E−9	NBO	TGG
58	M428Y	NBO	NBO	TAC
59	N434A	36.4E−9	NBO	GCC
60	N434C	2.5E−9	NBO	TGC
61	N434D	1.1E−9	NBO	GAC
62	N434E	6.1E−9	NBO	GAG
63	N434F	7.4E−9	NBO	TTC
64	N434G	7.4E−6	NBO	GGC
65	N434H	25.3E−9	NBO	CAC
66	N434I	NBO	NBO	ATC
67	N434K	31.7E−9	NBO	AAG
68	N434L	NBO	NBO	CTG
69	N434M	NBO	NBO	ATG
70	N434P	194.0E−9	NBO	CCC
71	N434Q	NBO	NBO	CAG
72	N434R	273.0E−9	NBO	AGG
73	N434S	114.0E−9	NBO	TCC
74	N434T	159.0E−9	NBO	ACC
75	N434V	6.7E−6	NBO	GTG
76	N434W	1.3E−9	NBO	TGG
77	N434Y	3.7E−9	NBO	TAC
78	D312A	17.6E−9	NBO	GCC
79	D312C	26.5E−9	NBO	TGC
80	D312E	42.5E−9	NBO	GAG
81	D312F	8.6E−9	NBO	TTC
82	D312G	38.4E−9	NBO	GGC
83	D312H	10.4E−9	NBO	CAC
84	D312I	15.2E−9	NBO	ATC
85	D312K	16.4E−9	NBO	AAG
86	D312L	5.7E−9	NBO	CTG
87	D312M	6.1E−9	NBO	ATG
88	D312N	25.3E−9	NBO	AAC
89	D312P	8.0E−9	NBO	CCC
90	D312Q	13.6E−9	NBO	CAG
91	D312R	9.0E−9	NBO	AGG
92	D312S	9.6E−9	NBO	TCC
93	D312T	9.9E−9	NBO	ACC
94	D312V	11.3E−9	NBO	GTG
95	D312W	13.2E−9	NBO	TGG
96	D312Y	9.4E−9	NBO	TAC
97	Y436A	13.3E−9	NBO	GCC
98	Y436C	12.5E−9	NBO	TGC
99	Y436D	11.6E−9	NBO	GAC
100	Y436E	13.4E−9	NBO	GAG
101	Y436F	9.3E−9	NBO	TTC
102	Y436G	51.7E−9	NBO	GGC
103	Y436H	NBO	NBO	CAC
104	Y436I	25.1E−9	NBO	ATC
105	Y436K	74.0E−9	NBO	AAG
106	Y436L	NBO	NBO	CTG
107	Y436M	5.5E−9	NBO	ATG
108	Y436N	65.5E−9	NBO	AAC
109	Y436P	NBO	NBO	CCC
110	Y436Q	9.1E−9	NBO	CAG
111	Y436R	7.7E−9	NBO	AGG
112	Y436S	169.0E−9	NBO	TCC
113	Y436T	36.2E−9	NBO	ACC
114	Y436V	6.7E−9	NBO	GTG
115	Y436W	28.2E−9	NBO	TGG
116	T286A	110.0E−9	NBO	GCC
117	T286C	84.4E−9	NBO	TGC
118	T286D	47.5E−9	NBO	GAC
119	T286E	35.5E−9	NBO	GAG
120	T286F	7.8E−9	NBO	TTC
121	T286G	18.2E−9	NBO	GGC
122	T286H	16.0E−9	NBO	CAC
123	T286I	15.8E−9	NBO	ATC
124	T286K	20.6E−9	NBO	AAG
125	T286L	14.0E−9	NBO	CTG
126	T286M	19.3E−9	NBO	ATG
127	T286N	NBO	NBO	AAC
128	T286P	6.9E−9	NBO	CCC
129	T286Q	7.7E−9	NBO	CAG
130	T286R	7.8E−9	NBO	AGG
131	T286S	17.1E−9	NBO	TCC
132	T286V	2.3E−9	NBO	GTG
133	T286W	15.4E−9	NBO	TGG
134	T286Y	14.9E−9	NBO	TAC
Mutants are numbered according to the EU Index as in Kabat.
NBO = No Binding Observed.

The results clearly show that mutations made at various positions have a marked effect on the affinity of the IgGs to equine FcRn.

Example 4

FcRn Binding Validations

Methods

The Fc regions of the equine FcRn and the four equine allotypes, IgG1, IgG4a, IgG4b, IgG7a and IgG7b were first designed using their respective CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by Deepmind was implemented to model the 3D structure of Equine FcRn and each of the wild-type (WT) and mutant constructs of each equine allotype.

Molecular Operating Environment (MOE) developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling. To analyze the difference in structures, the sequence-to-profile alignment algorithm uses a scoring algorithm to rank the sequence templates and scores higher than 85% ensure the selection of protein templates with physically realistic structures. The model was then optimized using the same pipeline and the structural stability of the models was verified using Ramachandran Plots, which checks the stereochemical quality of a protein structure.

Results

The method described above was performed on the Equine wild-type (WT) constructs and the following mutants in IgG1: M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, Q311A, Q311C, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, Q311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V, and Y436W. The positions for the mutational library are represented in a ball-and-stick form in FIG. 3.

The same procedure was also followed to investigate the following mutations in equine subclasses, IgG4 (IgG4a and IgG4b) and IgG7 (IgG7a and IgG7b): M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, K311A, K311C, K311D, K311E, K311F, K311G, K311H, K311I, K311K, K311L, K311M, K311N, K311P, K311R, K311S, K311T, K311V, K311W, K311Y, D312A, D312C, D312E, D312F, D312G, D312H, D312I, D312K, D312L, D312M, D312N, D312P, D312Q, D312R, D312S, D312T, D312V, D312W, D312Y, M428A, M428C, M428D, M428E, M428F, M428G, M428H, M428I, M428K, M428L, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428W, M428Y, N434A, N434C, N434D, N434E, N434F, N434G, N434H, N434I, N434K, N434L, N434M, N434P, N434Q, N434R, N434S, N434T, N434V, N434W, N434Y, Y436A, Y436C, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436P, Y436Q, Y436R, Y436S, Y436T, Y436V and Y436W. The positions for the mutational library are represented in a ball-and-stick form in FIG. 4 and FIG. 5.

An RMSD plot was generated to calculate the root mean square deviation of the WT structures, Equine IgG1, Equine-IgG4a, Equine-IgG4b, Equine-IgG7a and Equine IgG7b relative to each other (FIG. 6). An RMSD value of 2.0Å or lower is considered the standard for considering two structures to be alike. Results indicated that equine IgG4a construct was identical to the equine IgG4b allotype with average RMSD value for the structure being 0.44 Å (individual position RMSD in Table 4). Equine IgG7a construct was identical to the equine IgG7b allotype with an average RMSD value of 0.9 Å (individual position RMSD in Table 5). RMSDs at the positions where mutational scanning was performed was also noted in Tables 6 and 7 with values ranging from 0.226-0.997 Å.

TABLE 4
Root Mean Square Deviation (RMSD) comparisons of residues
in the protein models of WT Equine IgG4a and IgG4b.

	Residue in	Residue in
	EquinE-	EquinE-	RMSD
	IgG4a WT	IgG4b WT	({acute over (Å)})

	M252	M252	0.191
	T286	T286	0.476
	K311	K311	0.254
	D312	D312	0.220
	M428	M428	0.109
	N434	N434	0.059
	Y436	Y436	0.059

TABLE 5
Root Mean Square Deviation (RMSD) comparisons of residues
in the protein models of WT Equine IgG7a and IgG7b.

	Residue in	Residue in
	EquinE-	EquinE-	RMSD
	IgG7a WT	IgG7b WT	({acute over (Å)})

	M252	M252	0.859
	T286	T286	0.979
	K311	K311	0.783
	D312	D312	0.767
	M428	M428	0.765
	N434	N434	0.797
	Y436	Y436	0.780

TABLE 6
Root Mean Square Deviation (RMSD) comparisons of mutational
library in the protein models of equine IgG4a and IgG4b.

	Residue	Residue	RMSD
	in IgG4a	in IgG4b	({acute over (Å)})

	T286A	T286A	0.476
	T286C	T286C	0.476
	T286D	T286D	0.476
	T286E	T286E	0.476
	T286F	T286F	0.476
	T286G	T286G	0.476
	T286H	T286H	0.476
	T286I	T286I	0.476
	T286K	T286K	0.476
	T286L	T286L	0.476
	T286M	T286M	0.476
	T286N	T286N	0.476
	T286P	T286P	0.476
	T286Q	T286Q	0.476
	T286R	T286R	0.476
	T286S	T286S	0.476
	T286V	T286V	0.476
	T286W	T286W	0.476
	T286Y	T286Y	0.476
	K311A	K311A	0.254
	K311C	K311C	0.254
	K311D	K311D	0.254
	K311E	K311E	0.254
	K311F	K311F	0.254
	K311G	K311G	0.254
	K311H	K311H	0.254
	K311I	K311I	0.254
	K311Q	K311Q	0.254
	K311L	K311L	0.254
	K311M	K311M	0.254
	K311N	K311N	0.254
	K311P	K311P	0.254
	K311R	K311R	0.254
	K311S	K311S	0.254
	K311T	K311T	0.254
	K311V	K311V	0.254
	K311W	K311W	0.254
	K311Y	K311Y	0.254
	D312A	D312A	0.220
	D312C	D312C	0.220
	D312E	D312E	0.220
	D312F	D312F	0.220
	D312G	D312G	0.220
	D312H	D312H	0.220
	D312I	D312I	0.220
	D312K	D312K	0.220
	D312L	D312L	0.220
	D312M	D312M	0.220
	D312N	D312N	0.220
	D312P	D312P	0.220
	D312Q	D312Q	0.220
	D312R	D312R	0.220
	D312S	D312S	0.220
	D312T	D312T	0.220
	D312V	D312V	0.220
	D312W	D312W	0.220
	D312Y	D312Y	0.220
	M252C	M252C	0.191
	M252D	M252D	0.191
	M252E	M252E	0.191
	M252F	M252F	0.191
	M252G	M252G	0.191
	M252H	M252H	0.191
	M252I	M252I	0.191
	M252K	M252K	0.191
	M252L	M252L	0.191
	M252A	M252A	0.191
	M252N	M252N	0.191
	M252P	M252P	0.191
	M252Q	M252Q	0.191
	M252R	M252R	0.191
	M252S	M252S	0.191
	M252T	M252T	0.191
	M252V	M252V	0.191
	M252W	M252W	0.191
	M252Y	M252Y	0.191
	Y436A	Y436A	0.059
	Y436C	Y436C	0.059
	Y436D	Y436D	0.059
	Y436E	Y436E	0.059
	Y436F	Y436F	0.059
	Y436G	Y436G	0.059
	Y436H	Y436H	0.059
	Y436I	Y436I	0.059
	Y436K	Y436K	0.059
	Y436L	Y436L	0.059
	Y436M	Y436M	0.059
	Y436N	Y436N	0.059
	Y436P	Y436P	0.059
	Y436Q	Y436Q	0.059
	Y436R	Y436R	0.059
	Y436S	Y436S	0.059
	Y436T	Y436T	0.059
	Y436V	Y436V	0.059
	Y436W	Y436W	0.059
	M428C	M428C	0.109
	M428D	M428D	0.109
	M428E	M428E	0.109
	M428F	M428F	0.109
	M428G	M428G	0.109
	M428H	M428H	0.109
	M428I	M428I	0.109
	M428K	M428K	0.109
	M428L	M428L	0.109
	M428A	M428A	0.109
	M428N	M428N	0.109
	M428P	M428P	0.109
	M428Q	M428Q	0.109
	M428R	M428R	0.109
	M428S	M428S	0.109
	M428T	M428T	0.109
	M428V	M428V	0.109
	M428W	M428W	0.109
	M428Y	M428Y	0.109
	N434A	N434A	0.059
	N434C	N434C	0.059
	N434D	N434D	0.059
	N434E	N434E	0.059
	N434F	N434F	0.059
	N434G	N434G	0.059
	N434H	N434H	0.059
	N434I	N434I	0.059
	N434K	N434K	0.059
	N434L	N434L	0.059
	N434M	N434M	0.059
	N434Y	N434Y	0.059
	N434P	N434P	0.059
	N434Q	N434Q	0.059
	N434R	N434R	0.059
	N434S	N434S	0.059
	N434T	N434T	0.059
	N434V	N434V	0.059
	N434W	N434W	0.059

TABLE 7
Root Mean Square Deviation (RMSD) comparisons of mutational
library in the protein models of equine IgG7a and IgG7b.

	Residue	Residue	RMSD
	in IgG4a	in IgG4b	({acute over (Å)})

	T286A	T286A	0.979
	T286C	T286C	0.979
	T286D	T286D	0.979
	T286E	T286E	0.979
	T286F	T286F	0.979
	T286G	T286G	0.979
	T286H	T286H	0.979
	T286I	T286I	0.979
	T286K	T286K	0.979
	T286L	T286L	0.979
	T286M	T286M	0.979
	T286N	T286N	0.979
	T286P	T286P	0.979
	T286Q	T286Q	0.979
	T286R	T286R	0.979
	T286S	T286S	0.979
	T286V	T286V	0.979
	T286W	T286W	0.979
	T286Y	T286Y	0.979
	K311A	K311A	0.783
	K311C	K311C	0.783
	K311D	K311D	0.783
	K311E	K311E	0.783
	K311F	K311F	0.783
	K311G	K311G	0.783
	K311H	K311H	0.783
	K311I	K311I	0.783
	K311Q	K311Q	0.783
	K311L	K311L	0.783
	K311M	K311M	0.783
	K311N	K311N	0.783
	K311P	K311P	0.783
	K311R	K311R	0.783
	K311S	K311S	0.783
	K311T	K311T	0.783
	K311V	K311V	0.783
	K311W	K311W	0.783
	K311Y	K311Y	0.783
	D312A	D312A	0.767
	D312C	D312C	0.767
	D312E	D312E	0.767
	D312F	D312F	0.767
	D312G	D312G	0.767
	D312H	D312H	0.767
	D312I	D312I	0.767
	D312K	D312K	0.767
	D312L	D312L	0.767
	D312M	D312M	0.767
	D312N	D312N	0.767
	D312P	D312P	0.767
	D312Q	D312Q	0.767
	D312R	D312R	0.767
	D312S	D312S	0.767
	D312T	D312T	0.767
	D312V	D312V	0.767
	D312W	D312W	0.767
	D312Y	D312Y	0.767
	M252C	M252C	0.859
	M252D	M252D	0.859
	M252E	M252E	0.859
	M252F	M252F	0.859
	M252G	M252G	0.859
	M252H	M252H	0.859
	M252I	M252I	0.859
	M252K	M252K	0.859
	M252L	M252L	0.859
	M252A	M252A	0.859
	M252N	M252N	0.859
	M252P	M252P	0.859
	M252Q	M252Q	0.859
	M252R	M252R	0.859
	M252S	M252S	0.859
	M252T	M252T	0.859
	M252V	M252V	0.859
	M252W	M252W	0.859
	M252Y	M252Y	0.859
	Y436A	Y436A	0.780
	Y436C	Y436C	0.780
	Y436D	Y436D	0.780
	Y436E	Y436E	0.780
	Y436F	Y436F	0.780
	Y436G	Y436G	0.780
	Y436H	Y436H	0.780
	Y436I	Y436I	0.780
	Y436K	Y436K	0.780
	Y436L	Y436L	0.780
	Y436M	Y436M	0.780
	Y436N	Y436N	0.780
	Y436P	Y436P	0.780
	Y436Q	Y436Q	0.780
	Y436R	Y436R	0.780
	Y436S	Y436S	0.780
	Y436T	Y436T	0.780
	Y436V	Y436V	0.780
	Y436W	Y436W	0.780
	M428C	M428C	0.765
	M428D	M428D	0.765
	M428E	M428E	0.765
	M428F	M428F	0.765
	M428G	M428G	0.765
	M428H	M428H	0.765
	M428I	M428I	0.765
	M428K	M428K	0.765
	M428L	M428L	0.765
	M428A	M428A	0.765
	M428N	M428N	0.765
	M428P	M428P	0.765
	M428Q	M428Q	0.765
	M428R	M428R	0.765
	M428S	M428S	0.765
	M428T	M428T	0.765
	M428V	M428V	0.765
	M428W	M428W	0.765
	M428Y	M428Y	0.765
	N434A	N434A	0.797
	N434C	N434C	0.797
	N434D	N434D	0.797
	N434E	N434E	0.797
	N434F	N434F	0.797
	N434G	N434G	0.797
	N434H	N434H	0.797
	N434I	N434I	0.797
	N434K	N434K	0.797
	N434L	N434L	0.797
	N434M	N434M	0.797
	N434Y	N434Y	0.797
	N434P	N434P	0.797
	N434Q	N434Q	0.797
	N434R	N434R	0.797
	N434S	N434S	0.797
	N434T	N434T	0.797
	N434V	N434V	0.797
	N434W	N434W	0.797

Conclusion

Molecular modeling of all mutations across the two allotypes, IgG4a and IgG4b of equine backbones were performed and validated using MOE2019.0102 and AlphaFold2.2. Similarly, molecular modeling of all mutations across the two allotypes, IgG7a and IgG7b of equine backbones were performed and validated using MOE2019.0102 and AlphaFold2.2. The Fc fold and residue conformation between subclasses were shown to have RMSDs less than 2 Å, indicating that the protein structures have very high identity and therefore would function in a similar manner.

Having described preferred embodiments of the invention, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.